Volume 19 Number 9 September 2017 pp. 716–733 716 www.neoplasia.com

Swetha Tati†,1, John C. Fisk†,1, Julia Abdullah*, †, ‡,, Humanization of JAA-F11, a Highly † † Loukia Karacosta , Taylor Chrisikos*, , Specific Anti-Thomsen-Friedenreich Padraic Philbin*, Susan Morey*, Diala Ghazal†, In Vitro Fatma Zazala†, Joseph Jessee†, Sally Quataert†, Pancarcinoma Antibody and Stephen Koury*, David Moreno*, Jing Ying Eng*, Efficacy Analysis Vladislav V. Glinsky §, ¶, Olga V. Glinskii§, #, Muctarr Sesay**, Anthony W. Gebhard**, Karamveer Birthare**, James R. Olson†, †† and Kate Rittenhouse-Olson*, †

*Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, Buffalo, NY; †For-Robin, Inc., Buffalo, NY; ‡Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY; §Research Service, Harry S. Truman Memorial Veterans Hospital, Columbia, MO; ¶Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO; #Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO; **Goodwin Biotechnology, Inc., Plantation, FL; ††Department of Pharmacology and Toxicology, University at Buffalo, Buffalo, NY

Abstract JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine- conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model

Abbreviations: Ab, Antibodies; ADCC, Antibody-dependent cellular cytotoxicity; Ag, Friedenreich antigen; VH, Variable region heavy chain; VL, Variable region light Antigen; BLAST, Basic local alignment search tool; BSA, Bovine serum albumin; chain. CDC, Complement-dependent cytotoxicity; CDRs, Complementarity determining Address all correspondence to: Kate Rittenhouse-Olson, Department of Biotechnical regions; CHO, Chinese hamster ovary; CMV, Cytomegalovirus; DNA, Deoxyribo- and Clinical Laboratory Sciences, University at Buffalo, Buffalo, NY. nucleic acid; E. coli, Escherichia coli;EIA,Enzymeimmunoassay;ELISA, E-mail: [email protected] Enzyme-linked immunosorbent assay; FCS, Fetal calf serum; FRs, Framework 1 Dual first author. regions; Gal, Galactose; GalNAc, N-acetyl galactosamine; GlcNAc, N-acetyl Received 25 May 2017; Revised 19 July 2017; Accepted 24 July 2017 glucosamine; HC, Heavy chain; Ig, Immunoglobulin; IgG, Immunoglobulin G; © 2017 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This IgG1, Immunoglobulin G class 1; LC, Light chain; LDH, Lactate dehydrogenase; is an open access article under the CC BY-NC-ND license (http://creativecommons. mAb, monoclonal antibody; NeuAc, Sialic acid; OD, Optical density; PBS, Phosphate org/licenses/by-nc-nd/4.0/). buffered saline; PCR, Polymerase chain reaction; RNA, Ribonucleic acid; SD, 1476-5586 Standard deviation; SEM, Standard error of the means; TF-Ag, Thomsen- http://dx.doi.org/10.1016/j.neo.2017.07.001 Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 717

in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

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Background targeting ability that included 82% of triple negative breast cancers tested Thomsen-Friedenreich antigen (TF-Ag) is a carbohydrate pancarcinoma (TNBC) [28]. Furthermore, we recently demonstrated that JAA-F11 is antigen expressed on ~80% of human carcinomas including breast, lung, rapidly internalized by tumor cells within 1 hour [28]. This property can colon, bladder, ovary, prostate and stomach cancers, [1–10] but is cryptic be exploited to bring drugs or toxins into the cancer cells in a more on normal tissues. TF-Ag is also found on leukemia and lymphoma targeted way to reduce normal cell toxicity and increase tumor cell killing cells [11,12]. TF-Ag is expressed on tumor tissues due to changes in [30,31]. Altogether, these pre-clinical data obtained with JAA-F11 glycosylation in tumor cells which increases the expression of core suggest this antibody as a potential therapeutic agent for the treatment saccharide structures devoid of further glycosylation, as opposed to of cancer. normal cells where TF-Ag is hidden due to the addition of carbohydrate The fact that TF-Ag is associated with many human cancers, moieties [13–15]. TF-Ag is the disaccharide D-galactose-beta-(1–3)- including triple negative breast cancer, warrants its exploitation for N-acetyl galactosamine (Gal-β-(1–3)-GalNAc) which is alpha linked to a therapeutic uses. Murine antibodies, however, have limited therapeutic serine or a threonine residue on proteins [1]. The same disaccharide applications due to their immunogenicity, short serum half-lives, and structure can appear beta-linked on glycolipids in normal tissues, so this weak human effector functions [32–34]. Human immune response to linkage inclusion is important for tumor specificity [13]. Thus, the mouse JAA-F11 prevents its direct clinical use, so humanization is tumor restricted presence of this antigen makes this a promising target for necessary to decrease the immunogenicity of the mouse antibody and to cancer immunotherapy. In addition, TF-Ag plays a pivotal role in allow it to remain in circulation for a longer time. As humanization can metastasis [16–22] where tumor cell surface TF-Ag binds and causes affect specificity and affinity because the framework regions can have an up-regulation of Galectin-3 (Gal-3) expression and mobilization on the effect on conformation, understanding and conserving the initial surface of vascular endothelial cells. The TF-Ag and Gal-3 binding antibody structure and testing the humanized constructs is important. interaction causes the arrest of the tumor cell on the blood vessels followed This specificity is particularly important in our anti-carbohydrate by the endothelial integrin α3β1 stabilization/locking event, all steps antibody response since TF-Ag alpha is tumor associated while TF-Ag required for establishing metastasis [23]. Further evidence supporting the beta is on normal tissues. role of TF-Ag in tumor metastasis stems from clinical studies showing that Here we report the construction, production, and characterization of tumors that have greater metastatic activity were found to over-express two humanized variants of the mouse mAb JAA-F11. Humanization of TF-Ag [3–5,10]. Therefore, we hypothesized that therapy with an JAA-F11 was achieved by using a complementarity determining region anti-TF antibody could create a survival advantage for patients with (CDR) grafting process for the mouse VL and VH regions and TF-Agexpressingtumorsthroughdirect killing, antibody-linked drug subsequent construction into a full length human IgG1 antibody. The conjugate killing and/or by blocking tumor cell spread. antibody was produced in CHO-K1 cells after stable transfection with The murine monoclonal IgG3 antibody JAA-F11 [24] is highly two recombinant expression vectors. The purified hJAA-F11 antibodies specific in that it binds to the alpha-linked tumor-associated TF-Ag and were compared to the mouse [13,16,18,23,24,27,28,37] and chimeric not the beta-linked structure known to be localized on the surface of JAA-F11 in terms of specificity, relative affinity, and effector functions. normal tissues such as the kidney and natural killer (NK) cells [25–27]. The overall goal of this study is to generate a humanized monoclonal The in vivo specificity of JAA-F11 was observed using iodine 124-labeled antibody to the tumor antigen TF-Ag, which has high specificity, low JAA-F11 in the mouse 4T1 breast cancer model, the nude-xenograft immunogenicity and high biological efficacy. human breast cancer model (MDA-MB-231(triple negative)) and the SCID-xenograft human breast cancer model (with DU4475 triple Materials and Methods negative tumors) [27–29]. Notably, JAA-F11 did not significantly bind to the kidneys or other organs in the mice, which is a promising indicator Human use Ethics Statement of safety and specificity for future therapy and diagnostic imaging in Blood for cytotoxicity assays was obtained voluntarily from healthy humans. Conversely, the less specific anti-TF-Ag murine antibody, donors with approval (421397–4) granted by University at Buffalo Tru-Scint MAb 170H.82, which appears to bind to both the alpha- and Health Science Institutional Review Board. beta-linked TF-Ag, was withdrawn after Phase III clinical trials, possibly because despite the promising tumor imaging sensitivity observed, this Cells, Cell Lines, and Mammalian Expression Vectors antibody also bound normal kidney tissue [29]. Studies have shown that Adherent Chinese hamster ovary cells (CHO-K1; ATCC #CCL-61™, JAA-F11 blocks metastasis in an established in vivo 4T1 metastatic breast Manassas, VA) were maintained in Ham's F12 medium (Corning cancer model, as well as in in vitro and ex vivo models [16].Previousin Cellgro, Manassas, VA) supplemented with 10% fetal calf serum (FCS; vitro whole cell binding assays revealed that JAA-F11 targets Hyclone) at 37°C and 5% carbon dioxide (CO2) in humidified air. The approximately 80% of 41 breast cancer cell lines tested, regardless of tumorigenic human breast cancer cell line MDA-MB-231 was kindly estrogen receptor (ER), progesterone receptor (PR) or Her2 status, provided by Dr. Julian Gomez-Cambronero (Wright State University, 718 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

Ohio). The mouse breast cancer cell line 4T1 was obtained from Fred To choose an alternate human acceptor antibody FR, two separate Miller (University of Michigan). The breast cancer cell lines MDA-Kb2, Protein BLAST® (BLASTP) searches against the entire non-redundant SKBR-3, MDA-MB-453, MDA-MB-468, DU4475, HCC-1806 and human (Homo sapiens) Genbank database were performed for the heavy BT549 were obtained from the American Type Culture Collection and light chain variable regions of mouse JAA-F11. All non-Homo (ATCC breast cancer panel ATCC #30-4500K™, ATCC, Manassas, sapiens protein sequences, humanized antibodies and phage display VA) and maintained in recommended media. Lung cancer cell lines sequences were eliminated from the BLASTP results. The resulting top NCI-H446 (ATCC #HTB-171™), A549 (ATCC #CRM-CCL-185™), 10 most homologous sequences of each of the heavy and light chain NCI-H520 (ATCC #HTB-182™) and NCI-H460 (ATCC variable sequence of JAA-F11 were selected for the development of the #HTB-177™) were obtained from the American Type Culture human acceptor antibody framework for the humanized JAA-F11 Collection (ATCC, Manassas, VA) and maintained in recommended (hJAA-F11). At each spot in the framework of both the heavy and light media. Peripheral blood mononuclear cells (PBMCs) for variable region sequence the most frequently occurring human amino antibody-dependent cellular cytotoxicity (ADCC) assays were isolated acid among these top 10 selected human FR sequences was chosen to from whole blood obtained from healthy donors by density gradient replace the framework mouse amino acid except the 3 amino acids on centrifugation using Ficoll-Paque Plus (GE Healthcare) according to either side of the CDR were kept as mouse. These proposed hJAA-F11 manufacturer's instructions (SUNY University at Buffalo Health constructs were assessed for conformational effects on the binding site in Sciences IRB (HSIRB) Project: 421,397–4). collaboration with Dr. Andrew Gulick at the Hauptmann Woodward Two mammalian expression vectors, 6307 pAH and 6714 pAN, Institute. The heavy chains had a proposed replacement for alanine were kindly provided by Dr. Sherie L. Morrison (University of using arginine at position 72 that could potentially cause significant California, Los Angeles). The 6307 pAH vector contains the human steric interaction with surrounding amino acids. Similarly, a leucine at IgG1 heavy chain leader/constant region under the control of position 51 for the light chain was replaced with arginine which could human cytomegalovirus (CMV) promoter, and has an ampicillin result in steric clashes with surrounding amino acids side chains. These (Amp) and histidinol dehydrogenase (hisD) gene cassettes for conflicting substitutions were removed and replaced with the original selection; while the 6714 pAN vector carries a human kappa light mouse JAA-F11 framework residue, alanine 72 (VH) and leucine chain leader/constant region under the control of the human CMV 51 (VL). A chimeric JAA-F11 was also constructed in which the entire promoter and has a neomycin phosphotransferase (neoR) cassette mouse JAA-F11 variable region was attached to a human IgG1 constant for selection. Two other mammalian expression vectors were also region. This provided a baseline that should have the original specificity obtained from InvivoGen (San Diego, CA) for expression of human and affinity of the mouse antibody while having the human constant IgG1 heavy chains (pFUSE-ChIg-hIg1) and human kappa light chains region, to be used as a control for evaluating the humanized variant. (pFUSE2-CLIg-hK) which express resistance to zeomycin and blasticidin respectively. Construction of Humanized and Chimeric Antibody Expression Vectors Cloning and Sequencing of JAA-F11 Antibody Variable Regions The gene coding sequences for VH and VL chain variable regions of Cloning and sequencing of variable heavy (VH) and light (VL) genes of humanized and chimeric JAA-F11 were synthesized (Genscript, murine JAA-F11 mAb were performed after isolation of total RNA from Piscataway, NJ) with the inclusion of appropriate restriction enzyme JAA-F11 hybridoma cells using an RNeasy mini isolation kit (Qiagen, sites, and cloned into the pUC57 vector. To construct the humanized Louisville, KY) according to the manufacturer's instructions. First strand and chimeric heavy chain expression vectors, cloned humanized cDNA was synthesized via the Reverse Transcription System (Promega, JAA-F11 and chimeric heavy chain variable region DNA (VH) each Madison, WI) using oligo (dT) primers. Amplification of the VL gene was were excised from the pUC57 vector and ligated into the 6307 pAH performed using mixture of six leader region primers and a mixture of vector generating the humanized and chimeric heavy chain expression three J region primers [35]. The VH gene was amplified using gene vectors. The humanized and chimeric light chain vectors were obtained specific primers: 5′-TCTGGGGCTGAACTGGCAAAA-3′ (forward by excising the cloned humanized JAA-F11 and chimeric heavy chain primer) and 5′-CTTGGGTATTCTAGGCTCGATTCTC-3′ (reverse variable region DNA (VH) each from pUC57 vector and inserting into primer). All primers were synthesized by Invitrogen, Grand Island, NY. the 6714 pAN vector. Before transfecting into mammalian cells, all The PCR products were ligated into a pGEM-T Easy cloning vector expression vectors were verified by DNA sequencing. A similar strategy (Promega, Madison, WI) for sequencing analyses. was also used to clone into the heavy chain vector pFUSE-ChIg-hG1 and the light chain vector pFUSE2-CLIg-hK. Humanization of JAA-F11 TheCDRswereoptimizedusingtheKabat[36] and Chothia [37] Immunogenicity Prediction numbering systems, as well as crystal structure, glycan array and To determine the immunogenicity of the proposed humanized computational carbohydrate threading data [38].ThustheCDRsalso JAA-F11 variable region, a prediction was performed using the T20 included mouse JAA-F11 framework residues located 5–6 angstroms (Å) scoring method which was developed by Gao et al. [39] to calculate the from the antigen binding site obtained from the crystallography and “humanness” of monoclonal antibody variable region sequences. A blast computational carbohydrate threading data described in Tessier et al. of the variable region was performed against the T20 Cutoff Human [38],aswellastwocysteinesatpositionsL23andL88ofthelightvariable Database available online at http://abanalyzer.lakepharma.com/. The chain, which are involved in disulfide bonds. To select the most humanized antibody variable region was compared to the top 20 human mouse-like human germline sequence the IGMT database was searched Ab BLAST matches and scored for similarity to these sequences. The (http://www.imgt.org/) by removing the mouse CDRs from the T20 score for humanized antibody is obtained from the average of the sequence, and blasting the database for the most similar human germline percent identities of the top 20 matched human sequences. To be sequence FR. considered not immunogenic, T20 scores of the FR and CDR Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 719 sequences must be above 80, and T20 scores for the FR sequences only MA). Bound antibody was detected by incubation with alkaline must be above 85. Scores near or above these values are predicted to be phosphatase conjugated species specific anti-human IgG secondary of low immunogenicity [39]. antibody (Sigma, St. Louis, MO) and phosphatase substrate (Sigma, St. Louis, MO) and the absorbance reading was obtained at 490 nm. Stable Expression and Production of Humanized and Chimeric Each antibody was used at a concentration which would give an O.D. Antibodies of 1.0 in our ELISA as this was in the linear portion of the curve when For stable transfection into mammalian cells, an endotoxin-free plotting concentration versus O.D. and thus would be in an area plasmid isolation of humanized or chimeric heavy and light chain sensitive to inhibition. Thus H3L3 was used at a concentration of expression vectors was performed using PureYield Plasmid Midiprep 0.02 μg/ml, and H2aL2a was used at either 0.2 μg/ml or 0.15 μg/ml. System (Promega, Madison, Wisconsin) according to manufacturer's Chimeric was used at either 0.3 μg/ml or 0.25 μg/ml. The amount of instructions. Linearization was performed using the PvuI restriction mouse antibody required to inhibit the binding of 1 μg/mL enzyme (Promega, Madison, WI), and extracted usingtheS&SElutrap humanized or chimeric antibodies by 50% was determined. The electro-separation system (Schleicher & Schuell, Dassel, Germany) as per higher the amount of mouse antibody required for inhibition, the manufacturer's instructions. Five micrograms of each linearized heavy and higher the relative affinity of the antibody. The relative affinity of lightchainvectorwereaddedto5×106 CHO-K1 cells in cold Ham's each of the humanized antibodies and chimeric antibody was F12 medium in a total volume of 500 μL in a 0.4 cm electroporation compared using ANOVA analysis. cuvette (Bio-Rad, Hercules, CA). Following electroporation (Bio-Rad Gene Pulser, 960 μF and 250 mV), the cells were diluted with Immunofluorescent Staining Detection by Flow pre-warmed non-selective medium (Ham's F12 supplemented with 10% Single cell suspensions of in vitro propagated adherent cancer cell FCS) to 1 × 105 cells/mL and plated into 96-well flat bottom tissue lines, MDA-Kb2, MDA-MB-231, SKBR-3, MDA-MB-453, BT549, culture plates (BD Bioscience, San Jose, CA) at a density of 1 × 104 cells HCC-1806, A549, and HTB-171, were removed from tissue culture per well (200 μL/well). The transfected cells were incubated at 37°C and flasks using Cell Stripper solution (Corning Cat# 25–056-CL). Cells μ 5% CO2. Stable transfectants were selected with 700 g/mL G418 were washed twice with wash buffer (DPBS/2%BSA) and suspended at (Geneticin) and 5 mM of Histidinol by limiting dilution. The culture 1×107 cells/mL in assay buffer (DPBS/2%BSA/0.04% rat IgG (Sigma supernatants were analyzed by enzyme-linked immunoassay (ELISA) for Cat# 14131)) on ice bath for 30 minutes to block any sites that may antibody production using TF-Ag coated plates as previously described non-specifically bind IgG. All staining and wash steps were performed at [16]. The clones producing the highest amount of humanized or chimeric 4°C. After washing, 1 × 106 cells of each cell line were incubated for JAA-F11 were selected and expanded. The humanized and chimeric 60 minutes with 100 μL of either mouse JAA-F11 monoclonal antibodies were purified using a Protein A-Sepharose® 4B, Fast Flow antibody at 200 μg/mL or in assay buffer alone for controls for affinity column (Sigma-Aldrich, St Louis, MO). For continuous 60 minutes. Cells were washed twice to remove unbound antibody and production of hJAA-F11, the best selected stable clones of hJAA-F11 100 μL of biotinylated rat anti-IgG3 antibody (Biolegend, Cat# was put into a C2011 fibercell bioreactor (FiberCell Systems, Frederick, 406803) at 1.5 μg/ml was added for 30 minutes. After washing twice, MD) with DMEM and 10% CDM-HD protein free serum supplement 100 μL of streptavidin-conjugated PE (Biolegend, Cat# 405204) at (FiberCell). Antibody purified from bioreactor harvests was purified using 0.5 μg/ml was added for 60 minutes to detect the biotinylated anti-IgG a MabSelect Sure HiTrap column (GE Healthcare), followed by dialysis bound to JAA-F11 on the cancer cells. After the final two washes, cells against 50 mM sodium acetate, pH 5.5, and loaded onto a CaptoS were fixed with freshly made DPBS+ 2% paraformaldehyde (Electron column (GE Healthcare). Purified antibody was then eluted with a step Microscopy Sciences Cat# 15710) and read on a BD Fortessa™ flow gradient of sodium chloride. cytometer collecting a minimum of 104 cells. FCS data files were analyzed using TreeStar™FlowJo software. Humanized antibodies were Determination of Chemical Specificity by Printed Glycan Array diluted to 200 μg/mL in assay buffer and Goat anti-human IgG-biotin The chemical specificity of the humanized and chimeric JAA-F11 (Vector Labs, Cat# BA3000) at 1.5 μg/ml and stained as above. constructs were determined using the printed glycan array by the Parallel Flow Chamber Assay. The adhesion of MDA-MB-231 Consortium for Functional Glycomics, Emory, Ga. (http://www. cells to primary human pulmonary microvascular endothelial cells functionalglycomics.org/static/consortium/resources/resourcecoreh. (HPMEC, ScienCell Research Laboratories, Carlsbad, CA) was studied shtml) and was compared to data obtained previously for mouse in an in vitro parallel plate laminar flow chamber as follows. HPMEC JAA-F11 [27]. The method is described on the Consortium for were grown until 100% confluent in collagen I coated Ibidi μ-Slide Functional Glycomics website and is detailed by Blixt et al. [40]. The VI0.1 for 3 days under constant perfusion with complete growth media humanized JAA-F11, H2aL2a and H3L3, and chimeric constructs (ScienCell, Catalog #1001) at 1 μL/min. On the day of the experiment, were analyzed for reactivity with 610 glycans at 10 μg/ml. Data the HPMEC monolayer was exposed to increasing wall shear stress shown is for the glycans with binding to the antibody at or above 15% levels (3.0 dyn/cm2) by perfusing MDA-MB-231 single cell suspension of the binding RFUs seen with the TF-Ag-alpha-Sp8. (5×104 cells/ml) in warm RPMI media containing 0.75 mM Ca2+, Mg2+ and 10.0% FBS through a precision syringe infusion/withdrawal Determination of Relative Affinity pump KDS210 (KD Scientific, USA) for 30 minutes to activate The relative affinity binding of the humanized and chimeric endothelial cells [41]. Next, a single cell suspension of MDA-MB-231 JAA-F11 constructs to TF-Ag was performed using a competitive cells (5×104 cells/ml) was perfused for a 30 minutes period at inhibition ELISA with the mouse JAA-F11 antibody. Briefly, the physiological shear stress (0.4 dyn/cm2) in the presence of the antibody humanized or chimeric construct was incubated in the presence of tested or nonspecific human IgG as a control (10 mg/ml final different competing concentrations of mouse JAA-F11 on TF-Ag concentration), tumor cell interactions with HPMEC were observed coated 96-well plates Immunlon plates (Thermo Scientific, Milford, using an inverted phase contrast microscope Diavert, (Leitz Wetzlar, 720 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

Germany) and video recorded in at least 3 different fields for subsequent the media alone blank, representing effective secondary antibody alone offline frame-by-frame analysis. Tumor cells that remained in the same was subtracted from the wells containing antibody. The % position for at least 30 seconds were considered as stably adhered. The internalization was given as follows: amount of stably adhered cells was calculated for every 5 minute interval in each observation field. % internalization ° ° ° Lactate Dehydrogenase Release Cytotoxicity Assays. Antibody- ¼ ð1−ðabs 37 C sample−abs 37 CblankÞ=ðabs 4 C sample−abs 4 C blankÞ 100 dependent cellular cytotoxicity (ADCC) was determined using the lactate dehydrogenase (LDH) release assay (CytoTox 96; Promega, Controls cells with the secondary antibody conjugate followed by Madison, WI) with breast cancer cell lines according to manufacturer's substrate were subtracted from the test wells. Each assay was repeated 3 or protocol as previously described in Heimburg et al. [16] with some more times. The second method, used with one antibody to allow for modifications. In the ADCC assays, fresh PBMCs were used as effector visual confirmation of the enzyme immunoassay results, was an (E) cells, with human breast tumor target cells as target (T) cells at a immunofluorescence microscopy method to qualitatively visualize 100:1 E:T ratio. Either 50 or 200 μg/mL of humanized, mouse or internalized antibody and co-localization with lysosomal compartments, chimeric JAA-F11 antibodies in phenol-red free RPMI medium was and is described as follows. Mouse 4 T1 breast cancer cells (3 × 105 cells) added and incubated for 17 hours. The maximum release control (target were seeded on coverslips in the wells of two 6-well tissue culture plates cells and medium only and lysis solution) and volume control wells for 24 hours. Cells were then incubated with the humanized antibody (medium only) were performed. After incubation, supernatant was H2aL2a (1 μg/mL) diluted in serum-free RPMI 1640 at 4°C for collected, substrate added, and LDH release was measured at 490 nm. 20 minutes to allow antibody surface binding. Following the 20 minutes The release of LDH from lysed cells was used to quantify cytotoxicity incubation, antibody treated media was removed from the wells and and expressed as the percentage of lysis. Percent cytotoxicity was fresh serum-free RPMI media was added to one of the 6-well plates and calculated as 100 × [(E − SE)] / [(M − SL)], where E is the then placed at 37°C for 60 minutes to allow internalization. The other experimental well, SE is the spontaneous release without the antibody 6-well plate was considered as the 0 minute control plate where minimal control (target cells incubated with effector cells and PBS), M is the internalization takes place (maximum antibody surface binding) and this maximum release determined by the target cells lysed with 10× lysis plate was kept at 4°C. After the incubation steps at 37°C and 4°C solution and SL is spontaneous lysis of the target cell alone. In each respectively, coverslips in the 6 well plates were washed twice with experiment samples were performed in quadruplicate. The data is the ice-cold 5%BSA/PBS, rinsed once with 1× PBS and fixed with 4% average from experiments that were repeated three or more times except paraformaldehyde solution (Affymetrix) for 15 minutes at room where noted in the legend. The mouse antibody is used as a low to temperature. After three washes in 1× PBS, cells were permeabilized negative control as we had previously seen low to no ADCC with this with 0.1% Triton X-100, 0.1% sodium deoxycholate in PBS for mouse IgG3 antibody. 10 minutes at room temperature. After three rinses with 1× PBS, cells Cell Internalization Assays. To address whether the antibodies can were incubated in 5% BSA/PBS for 30 minutes at room temperature internalize into tumor cells for use as potential antibody-drug followed by an incubation with rabbit anti-lysosomal membrane protein conjugates, internalization after incubation at 37°C was evaluated 1 (LAMP1; Abcam, 24,170) antibody at 1 μg/mL in 5% BSA/PBS for using ELISA and was confirmed using immunofluorescence for one of 1 hour. The anti-lysosomal membrane protein was utilized as a marker the antibodies. A quantitative two-temperature enzyme immunoassay for lysosomal location that would indicate internalization. After three (ELISA), to measure loss of antibody surface binding after 37°C rinses with 1× PBS, the coverslips were incubated with anti-human incubation was performed as a measurement for internalization (as IgG-Alexa 647 and anti-rabbit IgG Alexa 488 (to detect LAMP 1) described in Ferguson et al. [28]). Briefly, Human breast cancer cell line secondary antibodies (Molecular Probes, Invitrogen), at a dilution of (MDA-MB-231 or BT549) was tested for mouse JAA-F11, H2aL2a 1:500 in 5% BSA/PBS for 1 hour at room temperature in the dark. and H3L3 internalization. Cell lines were grown to confluence in 6-well Finally, coverslips were rinsed three times with 1× PBS in the dark and cell culture plates. Media was decanted, and antibodies (JAA-F11 or mounted using the Slowfade Gold reagent (Molecular Probes, Life H2aL2a or H3L3) were added at 0.2 mg/ml in 3 wells, in the 3 control Technologies) with DAPI. Images were taken with an AxioImager wells 1 ml of media alone was added. One of the 6-well plates was fluorescence microscope (Zeiss) and analyzed using the AxioVision incubated at 37°C and other plate 4°C. One hour after incubation the Release 4.8.2 software. Images were captured using identical exposure 1 ml of media alone or antibodies in media were removed and the wells times for both the 37°C and 4°C conditions to avoid bias. Experiments were washed four times with appropriate cell culture media. After the were performed at least three independent times for the humanized last wash, 1 ml of freshly prepared 2% paraformaldehyde (methanol antibody. Color merging of the green for LAMP and the red for H2aL2a free) was added to each well and the plates were incubated for would indicate co-localization. 20 minutes at RT to fix the cells without permeabilizing the cell membrane. The plates were washed four times with media, followed by Preparation of the Antibody Drug Conjugate, H2aL2a-DM1 1 ml of either anti-mouse IgG (γ-chain-specific) alkaline phosphatase Briefly, 40 mg of hJAA-F11 human IgG1 Ab was reduced at one of secondary antibody (1:5000, Sigma, St. Louis, MO) or anti human IgG the two disulfide bonds at the hinge region, with tris(2-carboxyethyl) (Fab specific) alkaline phosphatase secondary antibody (1:1000, Sigma, phosphine (TCEP) at a 5:1 mole ratio of TCEP: IgG, for 60 minutes in a St. Louis, MO) was added and incubated at 37°C for 1 hour. Then 25°C stability chamber, and gently mixed using a rotary mixer. The plates were washed four times with media and 1 ml of p-nitrophenyl MC-DM1 linker payload was initially solubilized in DMSO and phosphate at 1 mg/ml was added and incubated for 1 hour at 37°C. At immediately added to partially TCEP-reduced hJAA-F11 human IgG1 1 hr., 200 microliters from each well was pipetted to a 96 well plate. Abata10:1moleratioDM1:IgG1togivea15%totalvolumeoforganic Absorbencies were read at 405 nm. Unreacted substrate served as the solvent. The sample was gently mixed for 60 minutes in a 25°C Stability blank. The triplicate wells were averaged, the average optical density of Chamber. After a 60 minute incubation, residual free thiols were Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 721 quenched with NEM at a ratio of 2:1 NEM: MC-DM1 for 30 min in a 15 mg/kg/mouse H2aL2a-DM1) and treatment was initiated after 25°C stability chamber while mixing with a rotary mixer. Next, the 24 hour of tumor cell inoculation. Each mouse was injected conjugate was purified/dialyzed overnight via four buffer changes at intraperitoneally (I.P.) on day 1, 4, 7 and then one injection per week 150:1 ratio of dialysis buffer:conjugate into 0.1 M Potassium Phosphate for 5 weeks. Tumor size was measured once every 3 days with a surgical pH 7.0. The conjugate was then characterized via HPLC-SEC for purity vernier caliper. At the end of the experiment (on day 50), the mice were with monitoring at wavelengths of 252 nm and 280 nm; drug to anesthetized with isoflurane gas and sacrificed. Tumors from each antibody ratio (DAR) was determined via HIC-HPLC as well as A252/ mouse were removed, measured with the digital caliper and weighed A280 ratio; SDS-PAGE (non-reducing) for purity and size; and A280 individually. Lungs, heart and liver were viewed for gross determination for protein concentration [42]. Each species was separated based on of metastasis for each mouse. the surface hydrophobicity on a Tosoh TSK gel Butyl-NPR column, For the second in vivo experiment, a few changes were made to the 4.6 mm I.D. × 3.5 cm, 2.5 μm (cat# 14947) at a flow rate of 1 ml/min above protocol; a pretreatment was done by adding 3 mg/mouse of and 20 μL injection of samples using a 1.5 M ammonium sulfate rabbit immunoglobulin I.P. to the MDA-MB-231 bearing mice on day gradient. DAR assignment was made based on elution time on column 6, and the antibody treatment of the mice did not begin until day 7, and and using a reference standard as a control. in addition fewer treatments were done, for a total of 6 weekly SEC-HPLC was used to assess the purity of the hJAA-F11 treatments for each group. The tumor bearing mice were divided H2aL2a-DM1 conjugate. The Tosoh Bioscience (G3000SW×1) column randomly into three groups (10 control treated with PBS, 10 mice was equilibrated with 100 mM sodium phosphate, pH 7.0, 100 mM treated with H2aL2a, 30 mg/kg/mouse, and 10 mice treated with sodium chloride, 15% isopropanol, on the Agilent 1100 HPLC column H2aL2a-DM1,15 mg/kg/mouse). Each mouse was injected intraperi- at a flow rate of 0.4 mL/min. Sample retention times were monitored at toneally on day 7 and weekly thereafter for 5 weeks. Tumor size was 252 nm and 280 nm. measured once every 3 days with a surgical vernier caliper. At the end of To determine purity of the final molecule, SDS-PAGE gel the experiment (on day 50), the mice were anesthetized with isoflurane electrophoresis was performed. Twenty microliters of each sample gas and sacrificed. Tumors from each mouse were removed, measured were mixed with 2× SDS-PAGE sample buffer (Novex, Waltham, MA) with the digital caliper and weighed individually. Lungs, heart and liver and loaded onto a 4–20% Tris-Glycine SDS-PAGE gel (Novex). Ten were viewed for gross determination of metastases for each mouse. micrograms of full-length undigested IgG1 were loaded as reference controls. The gel was electrophoresed for 2 h at 125 V, stained for 1 h in Statistical Analysis a G250 Coomassie stain (Bio-Rad, Hercules, CA), de-stained overnight Unpaired Student's t-test and ANOVA analysis with Dunnett's in high-purity water, and imaged using a Bio-Rad GS-800 densitometer. were utilized for statistical evaluation as described in the Figure Blue and Mark12 Molecular Weight Markers (Life Technologies, legends. P values b.05 were considered statistically significant. Carlsbad, CA) were loaded onto gel as molecular-weight reference standards. Immunohistochemistry Tumors from mice were removed on day 50 and placed into Z-fix for Cell Cytotoxicity In Vitro Assays 24 hours, followed by 70% ethanol. Tumors were then embedded in The effects of hJAA-F11-DM1 on tumor cell viability were assessed paraffin. Slides were deparaffinized by passing them through xylene, using Cell Titer-Glo reagent (Promega Corp.). Tumor cells (human 100% ethanol, 95% ethanol, 70% ethanol, followed by water. Slides breast cancer cell line: MDA-MB-231; human lung cancer cell lines: were washed twice with Tris-phosphate wash buffer and endogenous NCIH520; HTB171; A549) were plated in 96-well flat bottomed peroxidases were blocked with 3% hydrogen peroxide in methanol for plates (1000 cells per well) and allowed to adhere overnight at 37°C in a 15 minutes. Slides were then washed and endogenous biotin was blocked with an avidin-biotin blocking kit (Thermo Scientific). Following humidified atmosphere of 5% CO2. Cell culture medium was then removed and replaced by fresh culture medium containing different blocking with a 2.5% goat serum (Vector Labs), slides were incubated concentrations of H2aL2a-DM1, and the plates were incubated for a with either goat serum alone or biotinylated anti-human IgG antibody 5 day time period. After the 5 day time point, 10× Lysis buffer (Promega (Vector Labs) in goat serum for 30 minutes. Following washing, slides Corp.) was added to the appropriate positive control wells (for were treated using a Vectastain Elite ABC reagent (Vector Labs) for maximum lysis) and the plate was incubated at 37°C for 45 minutes, 30 minutes, then washed and developed with ImmPact DAB and then the plate was kept at RT for 30 minutes. Cell Titer-Glo chromogen (Vector Labs). Slides were counter stained with Modified reagent was added to the wells for 10 minute (2 minute on the orbital Harris Hematoxylin (Richard-Allan Scientific), dehydrated and then shaker) at RT and the luminescent signal was measured using a Synergy mounted. Slides were then scanned and images taken using an Aperio Neo2 Multi-Mode Reader (BioTek Instruments, Inc., VT, USA). IC50 Scan Scope GL (Leica BioSystems). was calculated using ic50.tk. Results Animal Efficacy In Vivo Studies The Designed Humanized JAA-F11 Antibody Constructs All animal experiments were performed using IACUC approved After the sequences of the mouse JAA-F11 variable regions were protocols in an AAALAC approved facility. Female SCID mice, verified, the CDRs for humanized JAA-F11 were defined so as to 6–8 weeks of age, were injected with 1 × 107 MDA-MB-231 cells maintain specificity and affinity for TF-Ag. The inclusive CDRs were subcutaneously, in a volume of 0.1 milliliters under the right upper defined according to both Kabat et al. [36] and Chothia et al. [37] abdominal nipple, under anesthesia (using Isoflurane gas) to allow based on sequence and structural variability, respectively. X-ray careful tumor cell placement. After inoculation, tumor bearing mice crystallography and computational carbohydrate threading studies on were divided randomly into four groups (9 control treated with PBS, mouse JAA-F11 [38] identified amino acid residues within 5 to 6 Å 8 mice treated with 30 mg/kg/mouse H2aL2a and 8 mice treated with from the binding site. These amino acids as well as cysteine residues at 722 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017 position L23 and L88 of the light chain that are important for hJAA-F11 Antibody Constructs Bind TF-Ag and Have the Same conformational structure were included in the final inclusive Alpha Linked TF-Ag Specificity as the Mouse JAA-F11 Antibody definition of the CDRs (Figure 1) used in JAA-F11 humanization. The first analysis of the new humanized antibody constructs was to To humanize the construct, human FR germline sequences were determine if the replacement of the FRs in JAA-F11 changed the selected by removing the CDR sequences from the mouse JAA-F11 exquisite specificity of the original mouse antibody for TF-Ag [27]. VH and VL sequences and blasted against the closest human The chemical specificity of the chimeric JAA-F11 and the hJAA-F11 sequences in the IGMT database (http://www.imgt.org/). This constructs were determined using a glycan array with 601–610 humanized JAA-F11 antibody is designated as hJAA-F11 H2aL2a different glycans by the Consortium for Functional Glycomics (Figure 2A). The other humanized construct (hJAA-F11 H3L3) used (CFG). These data were compared to data obtained previously for was prepared by selecting the top 10 most similar sequences in two mouse JAA-F11 [27]. The hJAA-F11 H2aL2a and H3L3 antibodies separate Protein BLAST® (BLASTP) searches against the entire showed similar binding as the chimeric antibody [43]. All antibodies non-redundant human (Homo sapiens) Genbank database for mouse have binding limited to only the Galβ1–3GalNAc-alpha (TF-Ag) JAA-F11 heavy and light chains as described in methods. At each spot linked structures (Figure 3), and lack of binding with α2–3 sialylated in the framework the most frequently occurring human amino acid TF-Ag structures (data in supplementary material). A critical point is among these top 10 selected human FR sequences was chosen to that the glycan array shows that out of 601–610 glycans, the replace the framework mouse amino acid, except that the 3 amino humanized hJAA-F11s and the chimeric F11 antibodies at 10 acids on either side of the CDR were kept as mouse (Figure 2A). micrograms/ml only bind TF-Ag-alpha linked and 2 other TF-Ag containing saccharide structures; similar results as shown previously Immunogenicity Prediction of Humanized and Chimeric for mouse JAA-F11 (Figure 3 and Tables 1 and 2). The lack of Antibodies binding with more than 600 negative saccharides on the array To assess the potential for the new hJAA-F11 constructs to elicit an indicates the strong potential targeting ability for these humanized anti-mouse response in humans, the VH and VL variable regions of antibodies. Important examples of closely related saccharides which hJAA-F11 H2aL2a and H3L3 were compared against the original do not bind are shown in Table 2 and include the TF related mouse sequence and scored using the T20 scoring system [39]. Using Galβ1–3GalNAc-beta linked structures. This indicates that the the T20 values of the hJAA-F11 H2aL2a and H3L3, and the chimeric antibody would bind to tumor tissues (coated with TF-Ag) and not to (Figure 2B), very low immunogenicity is expected in patients. This the central nervous system GM1 ganglioside, the asialo-GM1 of NK shows that the CDR-grafted antibodies are more human and expected cells, the GD1 of glycolipids or to asialo GM1 of peripheral nerve to be much less immunogenic than the chimeric antibody that was tissue, which display the beta-linked form (Table 2). The well-known produced by grafting the mouse variable region onto a human Fc elongation of TF-Ag on normal tissues is carried out by the enzyme region. β1–3 N-acetylglucosaminyl-transferase adding GlcNAcβ1–3 on the

Figure 1. Final CDRs for JAA-F11 heavy and light chain variable regions. Final CDRs as determined by Kabat (sequence) in red, Chothia (structure) in blue boxes, amino acids 5 Å from binding site (pink) or 6 Å from binding site (yellow). Black underlined = CDRs selected for humanization. In addition cysteine at position L23 and L88 (green) were maintained. (A) Heavy chain. (B) Light chain. Numbering is according to Kabat. Letters a, b, c, refers to insertions. Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 723

Figure 2. Amino acid sequence alignments of mouse (mJAA-F11) with humanized H2aL2a and H3L3 antibody and immunogenicity assessment of the H2aL2a and H3L3. A) Amino acid sequence alignments of mJAA-F11 heavy (top) and light (bottom) chain variable regions with variable regions of H2aL2a and H3L3 constructs. Green indicates the differences among the humanized antibodies and mJAA-F11. Alanine at position 71 (top) and Leucine at position 46 (bottom) shaded pink, indicate mouse residues that were retained to avoid steric clashes. The CDRs are shadedblue.NumberingisaccordingtoKabat.B)Assessmentofimmunogenicity of the H2aL2a and H3L3 constructs by using the scoring system developed by Gao et al. [39]. T20 score is used to measure “humanness” of monoclonal antibody variable region sequences; T20 scoreN 80 for FR and CDR sequences is not immunogenic in humans; T20 scoreN 85 for FR sequences only is not immunogenic in humans (see main text for details). The closer the number is to these cut-offs the less immunogenic the antibody. FR = framework; CDR = complementarity determining region.

Gal, forming GlcNAcβ1–3Galβ1–3GalNAcα-, or by a 2–3 sialyltrans- ferase forming Neu5Acα2–3Galβ1–3GalNAc (sialyl-TF) or with a second sialyltransferase forming Neu5Ac2–3Galβ1–3(Neu5Acα2–6) GalNAc (disialyl-TF). All of these structures show no binding to the mouse [27,38], chimeric or the humanized JAA-F11 constructs. Thus, normal tissue binding is not expected for JAA-F11 or the humanized hJAA-F11 antibodies. At 10 μg/ml the chimeric JAA-F11, and hJAA-F11 H2aL2a and H3L3 antibodies bind to an additional two

Table 1. Example RFU Graph Showing High Degree of Specificity of These Antibodies.

Figure 3. Binding of H3L3, H2aL2a and chimeric antibodies on a CFG Chart ID Saccharides of 601 Tested With ≥15% H3L3 Average H2aL2a Ave Chimeric Ave glycan array. The arrays contained 440 glycans (mouse) and 601–610 Binding to Any of the Antibodies RFU RFU RFU glycans (chimeric and humanized). Sample RFU vs. glycan binding is 139 Galb1–3GalNAcα-Sp8 13,343 21,619 13,860 shown for H3L3 since these graphs are so similar. Only TF-Ag and 3 137 Neu5Acβ2–6(Galβ1–3)GalNAcα-Sp8 7391 (55%) 25,576 (118%) 5390 (38%) or 4 related saccharides bound to the antibodies, and these are not 135 Neu5Acα2–6(Galβ1–3)GalNAcα-Sp8 1016 (8%) 6921 (32%) 3240 (23%) seen in normal human tissue. Saccharides shown if RFU ≥15% of Galβ1–3GalNAcα-Sp8 value for any construct. 724 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

Table 2. Four Similar TF-Ag Related Structures on Normal Tissues Table 3. Relative Affinity for TF-Ag: μg mJAA-F11 Required for 50% Inhibition of μgof The Glycan Array Data Showed that the Humanized hJAA-F11s and Chimeric JAA-F11 Antibodies do not hJAA-F11 (Chimeric, H2aL2a & H3L3) Antibody. The Average of at Least Three Independent Bind Galβ1–3GalNAc-Beta (β) Linked Structures and Also Common Elongation Structures on Normal Experiments are Shown ± Standard Deviation. Tissues. RFU Refers to Relative Fluorescence Unit. H2aL2a (μg) H3L3(μg) Chimeric(μg) Glycan Glycan Structure Linker H3L3 H2aL2a Chimeric Array # RFU RFU RFU 1.9 ± 0.3 33.3 ± 6.4**** 0.87 ± 0.2

a144 Galβ1–3GalNAcβ1-4(Neu5Acα2–3)Galβ14Glcβ- Sp0 10 7 -1 b145 Galβ1-3GalNAcβ1-4Galβ1-4Glcβ- Sp8 15 29 28 c88 GlcNAcβ1-3Galβ1-3GalNAcα- Sp8 19 49 78 IgG. The amount of mouse mJAA-F11 antibody that is required to d223 Neu5Acα2-3Galβ1-3GalNAcα- Sp8 11 22 109 inhibit 1 μg of hJAA-F11 or chimeric antibody to 50% is extrapolated All RFU on these saccharides are b1% of the reaction with the TF-Ag alpha disaccharide-Sp8. and taken as a measure of relative affinity. Inhibition curves are shown a Glycan #144 is ceramide ganglioside GM1 found on brain and red blood cells. in Figure 4. The higher the amount of mouse antibody required for b Glycan #145 is ceramide asialo-GM1 found on NK cells, kidneys, spleen, and regenerating respiratory epithelial cells. inhibition, the higher the relative affinity of the antibody. The results c Glycans # 88 and #223 are common elongation structures on normal tissues. are summarized in Table 3. This table shows the half maximal d Glycans # 88 and #223 are common elongation structures on normal tissues. inhibitory concentration of mouse JAA-F11 required to compete with 1 μg of this hJAA-F11 antibody. hJAA-F11 H3L3, and H2aL2a saccharides out of the 610 saccharides tested (Figure 3). However, antibody showed higher relative affinity to TF-Ag with a relative neither of the additional binding saccharides are expected to be a problem affinity of 33.32 +/− 6.4, and 1.95 +/− 0.3 relative to the mouse β – biologically for tumor targeting with hJAA-F11. The Neu5Ac 2 6 antibody, while the chimeric antibody with a half maximal inhibitory β – Gal 1 3GalNAc is not a natural structure in humans. The other concentration of 0.81 +/− 0.21 relative to the mouse antibody has a α – β – β structure, Neu5Ac 2 6(Gal 1 3)GalNAc , has only been reported in similar affinity as the mouse antibody as would be predicted since it respiratory mucin of a bronchiectasis patient [44], human midcycle has the same variable region (Figure 4B). Both humanized antibodies cervical mucin [45], human chorionic gonadotropin [46] and have a significantly higher relative affinity then the chimeric, P b .01. meconium, all that should not affect therapeutic use. Therefore, both humanized JAA-F11 constructs displayed better binding to TF-Ag than the original mouse antibody. Next, we wanted hJAA-F11 Constructs have Improved Relative Affinity to TF-Ag to investigate the biological activity of both of these humanized The relative binding affinities of the hJAA-F11 constructs and JAA-F11 antibodies. chimeric antibodies to TF-Ag were determined by comparing the ability of the mouse antibody to compete with the antibodies in an Flow Cytometric Analysis of TF-Ag Expression On Human enzyme immunoassay. In this assay, the humanized or chimeric Ab is Cancer Cell Lines with mJAA-F11, and hJAA-F11 Constructs mixed with serial dilutions of the mouse anti TF-Ag antibody H2aL2a and H3L3 (mJAA-F11). Binding of the human or chimeric anti-TF-Ag to the Multiple breast and lung cancer cell lines were examined by flow TF-Ag coated plate is measured using a species-specific anti-human cytometry using mJAA-F11 to determine relative TF-Ag expression

Figure 4. The humanized H3L3 has a higher affinity than H2aL2a antibody which has a higher affinity than chimeric antibody to TF-Ag-BSA. A) Graph shows relative affinity of H2aL2a and chimeric antibodies to TF-Ag as determined by ELISA. Each antibody was mixed with serial dilutions of mJAA-F11 and binding to the TF-Ag coated plate was measured by using a species-specific alkaline phosphatase anti-human IgG antibody. The amount of mJAA-F11 antibody required to inhibit 1 μg of each tested antibody (H3L3, H2aL2a, and chimeric antibodies) to 50% was extrapolated and is depicted in the table (B). The higher the amount of mouse antibody required for inhibition, the higher the relative affinity of the antibody. ANOVA analysis was performed on the relative affinity (IC50) values obtained for each antibody tested (as described above) and binding of H3L3 was significant at P b .001 (****). Humanized and chimeric antibodies used at a concentration which would yield an O.D. of 1.00 in the EIA when not inhibited. H3L3 used at ~10 times the H2aL2a concentration and ~15 times the chimeric concentration. Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 725 levels. The relative expression of TF-Ag for the human breast cancer cell (moderate expresser), and SKBR3, MDA-MB-468, MDA-MB-231, line MDA-MBA-231, is compared to other breast (Figure 5A) and lung MDA-MB-κb2, and Du4475 (low expressers) (Figure 5A). The two cell lines (Figure 5B). The breast cell lines in order of TF-Ag expression lung cancer cell lines tested are the adenocarcinoma line A549, and a are; BT549, and HCC1806 (both high expressers), MDA-MB-453 small cell lung cancer cell line, HTB-171 (Figure 5B) which show low

Figure 5. Flow cytometric analysis of TF-Ag Expression on Human Cancer cells. Flow histograms showing A) Relative TF-Ag expression levels on eight breast cancer cell lines BT549, HCC-1806, MDA-MB-453, SKBR-3, MDA-MB-468, MDA-MB-231, MDA-MB-kb2 and Du4475 detected with mJAA-F11 using rat anti-mouse IgG3-biotin/Streptavidin-PE. Solid black line is used to show the relative background with secondary antibody alone for MDA-MB-231. B) Relative TF-Ag expression levels on the breast line MDA-MB-231 compared to the lung cancer cell lines A549 and HTB-171, detected with mJAA-F11 using rat anti-mouse IgG3-biotin/Streptavidin-PE and C) binding of hJAA-F11 constructs H2aL2a and H3L3 to HTB-171 detected with Goat-anti-human IgG-biotin/Streptavidin-PE. Solid lines binding of JAA-F11 or hJAA-F11 antibody; dashed lines background binding with detection system only. 726 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017 and moderate expression respectively. Figure 5C, illustrates the relative Effector Functions of hJAA-F11 ability of hJAA-F11 constructs to bind HTB-171 cells. H3L3 binding is In vivo efficacy of antibody immunotherapy might involve many higher (MFI 1099) to TF-Ag on HTB-171 cells compared to H2aL2a different mechanisms including the immunologic effector functions binding (MFI 403) under the same conditions using a goat anti-human of the Ab. Therefore, the ability of the humanized antibody to carry IgG-biotin/Streptavidin-PE detection system. These results indicate out ADCC was determined using multiple human cancer cell lines that both hJAA-F11 antibodies can also be used to assess TF-Ag surface (Figure 7). At 200 μg/ml, H2aL2a was effective in ADCC killing expression on cell lines. against NCI-H520, BT549, HTB-171, and MDA-MB-231; while chimeric antibody showed little ADCC activity even at the 200 μg/ml hJAA-F11 Prevents MDA-MB-231 Cellular Adhesion to Normal concentration (b20% cytotoxicity) (Figure 7A). Mouse JAA-F11, as a Human Endothelial Cells negative or low antibody was also compared with H2aL2a and these TF-Ag is an important player in the adhesion of cancer cells to normal results are shown in Figure 7. Figure 7B shows that at 50 μg/ml, human cells, through the interaction of TF-Ag with the cellular lectin H3L3 facilitated statistically more ADCC than H2aL2a on all cell Galectin-3 [47,48]. This mechanism is considered one of the hallmarks lines tested. Even at the lowered concentration, H2aL2a still caused of metastasis as cancer cells bearing TF-Ag are thereby able to transverse lysis of ~20% of both the NCI-H520 and BT549 cells, whereas at from the primary tumor site and bind to the vasculature in a distal site to 50 μg/ml H3L3 caused N50% kill of NCIH520, close to 50% kill of initiate the formation of secondary tumors. An antibody that binds BT549 and MDA-MB-231, and ~30% kill of HTB-171 with the TF-Ag would feasibly be able to prevent metastasis by blocking the same effector to target ratio. This indicates that hJAA-F11 antibody TF-Ag interactions with vascular cells. To address this possibility, a constructs have potential for naked antibody immunotherapy. well-established parallel flow chamber assay was used to investigate the ability of hJAA-F11 to block metastasis-associated adhesion of human Humanized hJAA-F11 Internalizes Into Cancer Cells cancer cells to primary human microvascular endothelial cells Since antibodies can potentially be used to covalently carry drugs or [13,18,20,47,48]. This assay has been previously used to demonstrate toxins in the form of antibody-drug conjugates into cells, internalization that TF-Ag interactions with endothelium expressed galectin-3 are is an important aspect to analyze. Internalization of humanized and critical for mediating metastatic cell adhesion to endothelium under chimeric JAA-F11 into 4 T1 breast tumor cells was determined by two physiological flow conditions. Using this assay, TF-Ag positive metastatic methods, first an enzyme immunoassay with surface binding of human breast carcinoma cells MDA-MB-231 were passed over the antibody measured and compared after incubating the cells for 1 hour at monolayer of primary normal human pulmonary endothelial cells under either 4°C or 37°C and detection without a permeabilization step physiological conditions. The cells were treated with either control IgG, (Figure 8A). The second method was an immunofluorescence or anti-TF-Ag antibody (mouse JAA-F11 or hJAA-F11) and the cell microscopy method with surface staining for hJAA-F11 followed by adhesion was assessed by video microscopy. In the presence of control permeabilization and internal staining for hJAA-F11 and anti-LAMP-1 IgG, MDA-MB-231 strongly adhered to normal endothelial cells (lysosomal associated membrane protein, a lysosomal marker) and (Figure 6), however, in the presence of either JAA-F11 or hJAA-F11 DAPI (nuclear) staining (Figure 8B). Mouse JAA-F11 had previously H2aL2a, cancer cells were prevented from adhering to endothelial cells been shown to internalize within 1 hour at 37°C by the enzyme through the blocking of TF-Ag by either the original mouse or immunoassay method [28]. In the enzyme immunoassay, the majority humanized JAA-F11 antibody. Further, it appears that the humanized of mouse JAA-F11, chimeric, H2aL2a, and H3L3 antibodies were H2aL2a had improved blocking ability compared with the original shown to internalize into the human breast cancer line MDA-MB-231 mouse antibody. This is strong evidence that the humanized hJAA-F11 (Figure 8A), with H3L3 internalizing to the highest extent of the 3 can be used to block tumor cell metastasis-associated adhesive antibodies. In the immunofluorescence microscopy experiments, interactions and potentially prevent metastasis, which will be further H2aL2a is analyzed to give a visual conformation of the enzyme investigated in future studies. immunoassay results. In agreement with the enzyme immunoassay, the majority of H2aL2a antibody showed internalization and co-localization with the lysosomal protein marker, LAMP-1, in the MDA-MB-231 cell line (Figure 8B). Therefore, both assays confirm that these antibodies internalize.

Antibody–Drug Conjugate (ADC) Characterization and In Vitro Cytotoxicity Many current targeted therapies now rely on conjugating potent (but relatively nonspecific) anti-cancer drugs directly to a specificity factor in the form of an antibody. Therefore, as proof-of-principle for hJAA-F11 as the antibody in an ADC, the microtubulin inhibitor ′ ′ DM1 (N2 -Deacetyl-N2 -(3-mercapto-1-oxopropyl) maytansine) was conjugated to hJAA-F11 H2aL2a with a pH sensitive linker that would release the payload upon internalization and acidification Figure 6. Anti-metastatic effect of mouse JAA-F11 and hJAA-F11 in within the lysosome. The site directed conjugation of MC-DM1 an in vitro model. Graph comparing humanized JAA-F11 H2aL2a, and 2′ 2′ JAA-F11 effect on adhesion of MDA-MB-231 to primary pulmonary (maleimidocaproyl-N -Deacetyl-N -(3-mercapto-1-oxopropyl) microvascular cells. ANOVA analysis differences are highly signifi- maytansine) to partially TCEP (tris(2-carboxyethyl) phosphine), cant (P b .0001). Adherent cells are those that stay attached to the which reduces solvent exposed disulfides, reduced hJAA-F11 Ab, microvascular cells for at least 30 seconds. resulting in an ADC with an average DAR of 3.54 as determined via Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 727

mole ratio of DM1:Ab by A252/A280 and a DAR of 2.12 as determined by HIC-HPLC peak integration, with the predominant species being a DAR of 2 as determined via HIC-HPLC (Figure 9) [42]. HIC-HPLC was used to determine the DAR of the hJAA-F11 DM1 conjugate based on the elution time of each individual species contained within the conjugate [27]. Furthermore, the hJAA-F11 conjugate was greater than 93% pure as determined via SEC-HPLC. Using an in vitro cell culture approach, the sensitivity of various human cancer cell lines to DM1 conjugated hJAA-F11 was analyzed. Several well studied triple negative human breast cancer (TNBC) lines (MDA-MB-231, MDA-MB-468, and MDA-MB-453) were treated. Increasing amounts of hJAA-F11-DM1 was titrated with each cell line and after 5 days, the cell viability was determined. All three TNBCs showed sensitivity to hJAA-F11-DM1, with MDA-MB-468 having an IC50 of 0.01 μg/mL hJAA-F11-DM1, MDA-MB-453 with an IC50 of 1.2 μg/mL and MDA-MB-231, the μ least sensitive, having the highest IC50 at 2.1 g/mL (Figure 9B). A similarexperimentusingonlynakedhJAA-F11showedno discernable killing of MDA-MB-231 (data not shown), indicating that the specific delivery of DM1 through hJAA-F11 was responsible for cancer cell killing. TF-Ag is also present on many carcinoma types, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). To determine the feasibility of using hJAA-F11-DM1 for lung cancer therapy, we tested TF-Ag positive lung cancer lines SCLC HTB-171(NCI-H446) [49,50] and the TF-Ag positive NSCLC squamous cell carcinoma NCI-H520 (HTB-182) [51] and the NSCLC adenocarcinoma A549 [52,53]. While all the lines showed sensitivity to hJAA-F11-DM1, the medium μ TF-Ag expressing HTB-171 was more sensitive (IC50 0.34 g/mL), followed by NCI-H520 (IC50 1.35) and the lower TF-Ag expressing A549, showed sensitivity similar to the breast cancer line μ MDA-MB-231 (IC50 2.1 g/mL) (Figure 9C). HIC-HPLC was used to determine the DAR of the hJAA-F11 DM1 conjugate based on the elution time of each individual species contained within the conjugate [27]. In conclusion, hJAA-F11-DM1 also shows promise as a lung cancer therapy, which will be focused on more fully in a future study.

In Vivo Efficacy of hJAA-F11 and hJAA-F11-DM1 As hJAA-F11-DM1 showed the ability to kill the human TNBC cell line MDA-MB-231 in culture, the ability of drug conjugated hJAA-F11 to kill tumor cells in the in vivo mouse xenograft model was addressed (Figure 10). Even though this cell line has low TF-Ag expression (Figure 6), it was chosen for the in vivo analysis because it Figure 7. ADCC activity versus TF-Ag positive breast and lung cancer is a human triple negative model, and is one of the most widely used cell lines by 7a) H2aL2a and chimeric antibodies at 200 μg/ml, and breast xenograft models. In the first in vivo experiment (Figure 10), 7b) H3L3 and H2aL2a Ab at 50 μg/ml. A) 200 μg/ml of the H2aL2a and mice were inoculated with MDA-MB-231 cells and 24 hours later chimeric were used in ADCC assays against several human cancer cell treatment was initiated, including a control group treated with lines. BC indicates a breast cancer line, while LC indicates a lung PBS, and experimental groups treated with hJAA-F11 H2aL2a at cancer line. H2aLa was most effective in ADCC against NCI-H520, then in decreasing amounts towards BT549, HTB-171, and MDA-MB-231, 30 mg/kg, and a group treated with H2aL2a-DM1 at 15 mg/kg. and the chimeric antibody showed little ADCC even at this 200 μg/ml Treatments were administered i.p. on day 1, 4, 7 and thereafter once concentration (P b .05) B) 50 μg/ml of either H2aL2a or H3L3 were per week for 5 weeks. Tumor size was measured once every 3 days, and used in ADCC assays against several human cancer cell lines. on day 50, the mice were sacrificed, and tumors were removed, measured C) 200 μg/ml concentrations of either H2aL2a were added to BT549 and weighed. Figure 10 panels A and B show the early effect of both cells. The mouse JAA-F11, effectively a negative control antibody did 30 mg/kg hJAA-F11 (statistically smaller at day 30) and 15 mg/kg μ not show appreciable ADCC even at 200 g/ml with the BT549 cells. In hJAA-F11-DM1 conjugate (statistically significant from day 25–30, the HTB-171 and MDA-MB-231 cell lines with H2aL2a, there was no appreciable ADCC activity above background and was not graphed. and day 42- day 50). Panels C and D show the tumor volume data to • indicates significance differences in one representative experiment. day 50 and the tumor weight data at day 50, respectively. The ** indicates significance in two of three independent experiments. hJAA-F11-DM1 conjugate causes a statistically significant difference in *** indicates significance across all three independent experiments. both of these measurements at day 50. 728 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

Figure 8. Internalization of H3L3 and H2aL2a antibodies in 4 T1 and MDA-MB-231 cells. A) Internalization by EIA detection of antibody surface binding following incubation of cells with antibody at 4°C (no internalization) and 37°C degrees (to induce internalization). All antibodies displayed internalization, where H2aL2a displayed significantly more internalization than chimeric (*, P b .05) and H3L3 showing significantly more internalization compared to all antibodies (**, P b .05). Bars represent mean ± S.E. (n = 3 experiments). B) Representative images showing immunofluorescent staining of Ab H2aL2a and the lysosomal protein marker, LAMP-1 (green). Top panels MDA-MB-231 cells were pre-incubated with antibody (1ug/mL) for 20 min at 4°C. Lower panels after at 37°C for 60 min. Merged images confirm internalization of H2aL2a and lysosomal co-localization with LAMP-1 in merged images (arrows). Total magnification was 40×.

The above experiment was repeated with two modifications, a constructs have great potential to be well tolerated in adjunct cancer pretreatment with 3 mg/mouse of rabbit IgG was utilized to block Fc therapy. Humanized JAA-F11antibodies can also be developed for receptors in the SCID mice, and the first injection of antibody was tumor localization, since TF-Ag is not secreted and thus not present delayed to day 7 after tumor implantation and continued weekly. In in the serum. This has been supported by mouse 124-I-JAA-F11 Figure 11A, both H2aL2a and H2aL2a-DM1 inhibited growth imaging studies in syngeneic and xenogeneic tumor models in mice, statistically as measured by tumor volume from day 38 to the where long tumor retention time was observed [27]. completion of the study. The tumor volumes of both the naked H2aL2a What makes the patented JAA-F11 antibody unique for this antigen is and the H2aL2a–DM1 conjugate treated mice were significantly its high degree of specificity, in that it reacts to the unsubstituted decreased relative to the control treated mice at time points indicated by Galβ1–3GalNAc alpha linked to serine (the true tumor TF-Ag), and not the asterisks. At day 50, tumors were removed and while the volumes of to the Galb1–3GalNAc beta structure in glycolipids which is on some the removed tumors remained significantly different, tumor weight only normal tissues including the kidney. By developing humanized JAA-F11 approached significance (P = .0506) in the H2aL2a-DM1 group constructs through careful inclusive use of Kabat and Chothia guidelines (Figure 11B). Figure 12 shows that after I.P. injection hJAA-F11 can be as well as crystallography and computational carbohydrate threading detected in the tumor of a SCID mouse by IHC after hJAA-F11 (an innovative additional step) to determine the CDRs, we maintained treatment confirming the role of the humanized antibody in the efficacy or enhanced antibody specificity and affinity (Figures 3 and 4 and data. Even though this xenograft model system was not optimized for Tables 1-3). The affinity and the specificity of the humanized antibody hJAA-F11 therapy since MDA-MB-231 has low to moderate TF-Ag for the antigen were fully retained or improved when compared to its expression, and the H2aL2a is of lower affinity than the more recently mouse and chimeric counterpart. Through use of the top 10 most similar acquired H3L3, H2aL2a- DM1 and naked hJAA-F11 antibody have human antibodies in the construction of the humanized antibodies, we potential as adjunct therapy for TNBC. were able to obtain two hJAA-F11 constructs (H2aL2a and H3L3) with T20 scores [39] that indicate little to no immunogenicity (Figure 2B), Discussion suggesting promising immunotherapeutic properties. The humanized TF-Ag, the disaccharide tumor antigen targeted by JAA-F11, has hJAA-F11 antibodies H2aL2a and H3L3 showed promising ADCC been studied for many years, and there is strong evidence concerning properties, significantly inducing breast and lung cancer cell killing its tumor associated presence on ~80% of carcinomas and lack of in vitro when compared to the chimeric antibody and the mouse expression in normal tissues [1–10]. It is an adhesion molecule related antibody (Figure 7). This ADCC improvement in the switch from a to invasiveness, so use of an antibody targeting this molecule has mouse IgG3 to a human IgG1 is similar to that seen by Cheung et al. potential as an immunotherapeutic, as an antibody-drug conjugate for with their anti-GD2 antibody [57]. In addition, hJAA-F11 H2aL2a was direct tumor cell killing and for blocking metastasis. Another unique shown to reduce tumor growth in a mouse xenograft human triple feature of TF-Ag as a target is that patients have naturally occurring negative breast cancer model (Figures 10 and 11). antibodies to TF-Ag, and these antibody levels are positively Importantly, the chimeric and humanized hJAA-F11 H2aL2a and correlated with prognosis without causing any autoimmunity H3L3 antibodies were also shown to internalize rapidly, with [1,54–56] indicating low potential toxicity. Thus, hJAA-F11 68–95% of the antibody being internalized within 1 hour (Figure 8), Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 729

Figure 9. Characterization of Maytansine (DM1) Conjugate of H2aL2a. A) H2aL2a-DM1 conjugate production. HIC-HPLC Chromatogram of H2aL2a Ab and H2aL2a-DM1 conjugate at A280. B) In vitro cell efficacy against human breast cancer (BC) cell lines using H2aL2a-DM1. C) In vitro efficacy against human lung cancer cell lines using H2aL2a-DM1. NSCLC is a non-small cell lung cancer and SCLC is a small cell lung cancer. B) & C) DM1 conjugated H2aL2a antibody showed enhanced anti proliferative activity on TF-Ag positive breast and lung cancer cell lines. All experiments were carried out to Day 5 of treatment and cell viability detected using a Cell-Glo reagent. (IC50 values, μg/mL H2aL2a-DM1). comparable or improved when compared to the mouse JAA-F11 cancer cell lines that showed varying TF-Ag expression (Figures 6 and 9B). antibody [28]. This observation lead to construction of our proof of Initial in vivo efficacy studies also demonstrated that hJAA-F11-DM1 principle ADC, hJAA-F11-DM1 conjugate which was shown to have conjugate reduced tumor growth (volume and weight) in a mouse potent in vitro cytotoxic activity in several human breast and lung xenograft human triple negative breast cancer model (Figures 10 and 11). 730 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

Figure 10. In vivo efficacy of H2aL2a and H2aL2a conjugated DM1 in breast tumor model treatment beginning 24 hours after tumor implantation. A) 1 × 107 MDA-MB- 231 cells were injected into female SCID mice intra-mammary gland on day 0. Antibody treatment began on day 1 and animals were given i.p. injection of PBS, naked H2aL2a (30 mg/kg), or H2aL2a-DM1 (15 mg/kg). Arrows indicate injection days. n = mice per group. Tumor growth was monitored for 50 days using caliper. A shows the first 30 days of treatment, B is the full 50 day experiment. Two-way ANOVA Analysis of PBS versus H2aL2a-DM1 *P = .05, **P = .01, ****P b .001. C) Bar graphs representing the mean tumor weights ± S.E in the control and antibody treatment groups of tumors removed at day 50. * Unpaired t test was used to analyze the PBS control versus H2aL2a-DM1.

Together, the results suggest that hJAA-F11 H2aL2a, both conjugated to metastatic breast tumors and plays a major role in the processes associated DM1 and the naked antibody have potential as adjunct therapy for with tumor metastasis [16–22], the critical new finding that our TNBC, even though this xenograft model was not optimized, since constructs are internalized by tumors cells makes our humanized antibody MDA-MB-231 is a low to moderate TF-Ag expresser and the H2aL2a is a an ideal candidate for antibody-drug conjugate therapy specifically for lower affinity hJAA-F11 construct than the more recently acquired H3L3 metastatic breast cancers. construct. Currently, the ADCs, Kadcyla and Adcetris, have successfully shown efficacy in cancer patients. Adcetris contains an antibody which Conclusion targets CD-30 that is conjugated to monomethyl auristatin, and is In conclusion, this study highlights the therapeutic potential of our applicable in Hodgkin’s lymphoma and systemic anaplastic large cell hJAA-F11 H2aL2a and H3L3 antibodies. Further development and lymphoma [31]. Kadcyla contains antibody that is conjugated to a optimization of these humanized anti-TF-Ag antibodies could lead to microtubulin inhibitor, and is applicable to the 20% of breast cancers that their use in direct immunotherapy and use as an antibody-drug are Her-2 over-expressers [58]. However, whereas Kadcyla is applicable in conjugate, in conjunction with other approaches for treating breast, only about 25% of breast cancer patients, based on preliminary testing, lung, and other cancers. Importantly, hJAA-F11 holds great promise, JAA-F11 and our humanized JAA-F11 antibodies should be applicable in especially for triple-negative breast cancers for which there are no up to 80% of breast cancers. Given that TF-Ag is highly expressed on current successful targeted therapies. Neoplasia Vol. 19, No. 9, 2017 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. 731

Figure 11. In vivo efficacy of H2aL2a and H2aL2a conjugated DM1 in breast tumor model treatment with antibody treatment beginning at day seven after tumor implantation. A) 1 × 107 MDA-MB-231 cells were injected into female SCID mice intra-mammary gland on day 0. Antibody treatment began on day 7 and animals were given i.p. injection of PBS, H2aL2a (30 mg/kg), or H2aL2a-DM1 (15 mg/kg). Mice were injected once a week for 6 weeks. Arrows indicate injection days. n = 10 mice per group. Tumor growth was monitored for 50 days. ANOVA Analysis of PBS versus H2aL2a-DM1, PBS versus 30 mg/kg H2aL2a (*P =.05,**P =.01,****P b .001). B) Bar graphs representing the mean tumor weights ± S.E in the control and antibody treatment groups of tumors removed at day 50. * Unpaired t test was used to analyze the PBS control versus H2aL2a-DM1. Treatment groups were approaching significance when tumor weights were compared.

Competing Interests molecular aspects of this paper, as well as the IC50 experiments, KRO owns the company, For-Robin, which has licensed the JAA-F11 and LK and TC did the original biological experiments; JA, JYE, and hJAA-F11 antibodies from the University at Buffalo. The and SK worked on early molecular aspects of the paper; JA also University at Buffalo and KRO have the patent for the mouse performed biological experiments, SMM performed the cell culture; antibody. KRO does not receive any salary or remuneration from the FZ, JJ, and DG performed the relative affinity experiments; DG company at this time or at any time in the past. The University of also assisted with the animal experiments; LK and DM performed Buffalo, KRO, JA, JYE and SK have applied for a patent for the some internalization experiments. VVG and OVG performed the humanized antibody. Except where noted there are no other flow cell experiment; SQ designed and analyzed the flow cytometry; competing interests for any of the authors. ST,JF,JA,LK,JO,andKROallpreparedthedraftofthe manuscript. MS, AG, and KG prepared and analyzed the DM-1 Author Contributions conjugate; JO and KRO conceived this study and participated in its ST performed the animal experiments, the internalization experi- design and coordination. All authors read and approved the final ments, the ADCC and the flow experiments, JF worked on all manuscript.

Figure 12. Immunohistochemistry to show tumor localization of H2aL2a after treatment of tumor bearing mice. The tumors from mice in the treatment study shown in Figure 11, injected with either PBS (A) or H2aL2a (B) were fixed, embedded and sectioned and stained using immunohistochemistry for the presence of human IgG in the tumor using anti-human secondary antibody. This shows the presence of H2aL2a binding to the tumor, while PBS indicates minimal background staining alone. 732 Humanization and Preclinical Efficacy of JAA-F11, a specific Anti-TF-Ag-α antibody Tati et al. Neoplasia Vol. 19, No. 9, 2017

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